Adjuvant properties of poly (amidoamine) dendrimers

ABSTRACT

A new method of adjuvanting a variety of materials has been developed. Starburst dendrimers, primarily poly(amidoamine) starburst dendrimers, can be used as an adjuvant for Influenza antigen and similar materials. Mid-Generation dendrimers are preferred and yield high antibody titer levels with reduced antigen dosage.

The present invention concerns the use of poly(amidoamine) dendrimers asadjuvants for enhancing the immune response to a variety of antigens.These poly(amidoamine) dendrimers are particularly useful in vaccinesbecause lower doses of the antigen can be used than in unadjuvantedvaccines.

Dendrimers, as the term is used herein, are a class of polymers oftencalled starburst polymers because of their shape. These dendrimers havea molecular architecture with an initiator core, interior layers (or"generations") of repeating units regularly attached to this initiatorcore, and an exterior surface of terminal groups attached to theoutermost generation. These starburst polymers are radially symmetricaland have a branched or tree-like structure. The number of generationscan be controlled by the conditions of manufacture, leading to differentsize molecules having different numbers of terminal groups. U.S. Pat.No. 4,587,329 entitled Dense Star Polymers Having Two DimensionalMolecular Diameter, issued May 6, 1986 to the Dow Chemical Company, thedisclosure of which is incorporated by reference, describes thesestarburst dendrimers and methods of their manufacture. These starburstdendrimers can be made to exact, repeatable molecular weights with thesame number of functional groups on each dendrimer. These functionalgroups can react with a material to be carried, such as a pharmaceuticalor agricultural product, or the material to be carried can be associatedwith this dendrimer in a non-reactive manner.

The family of dendrimers useful in the present invention are based on anamidoamine repeat structure, forming what are known as poly(amidoamine)dendrimers ("PAMAM"). PAMAM dendrimers are grown from an aminecontaining core structure such as ammonia, ethylene diamine, or thelike. Normally ethylene diamine is used as the core or initiator of thereaction. FIG. 1 shows the basic synthesis for the first threegenerations of these PAMAM starburst dendrimers. Ethylene diamine (EDA)is reacted with methyl acrylate under control conditions such that aMichael addition of one molecule of EDA to four molecules of methylacrylate occurs. This forms the initiator or core adduct. Following theremoval of excess methyl acrylate, the core adduct is reacted with anexcess of EDA to form a 0 generation molecule having four amidoaminegroups. The excess EDA is removed and the 0 generation molecule can bereacted with methyl acrylate in another Michael addition reaction toform a first generation molecule containing eight primary amine groups.A continuation of this stepwise procedure forms the other generations insequence. Table 1 list the molecular weight and number of primary aminegroups for the generation 0-10 PAMAM starburst dendrimers.

                  TABLE 1                                                         ______________________________________                                        Generation   MW      Primary Amino Groups                                     ______________________________________                                        0            517     4                                                        1            1430    8                                                        2            3256    16                                                       3            6909    32                                                       4            14215   64                                                       5            28826   128                                                      6            58048   256                                                      7            116493  512                                                      8            233383  1024                                                     9            467126  2048                                                     10           934787  4096                                                     ______________________________________                                    

As noted, these starburst polymers have been known to be useful with avariety of pharmaceuticals and agricultural products. In addition, ithas been theorized that they may have immuno-potentiating response. SeeU.S. Pat. No. 5,338,532, entitled: Starburst Adjuvants, issued Aug.16,1994, to the Dow Chemical Company, the disclosure of which is alsoincorporated herein by reference. However, there are no tests shown ofthis proposed immuno-potentiating effect.

Vaccines are composed of antigens which, when administeredsystematically, predominately stimulate an antibody response. Adjuvantsare materials which enhance the ability of the antigen or vaccine togenerate an antibody response or cytotoxic T cell response in vivo.Adjuvants are necessary for a number of antigens since the immuneresponse maybe too low without the adjuvant to provide a protectivelevel of antibody. Adjuvants enhance the immune response of the body,yielding higher titers and requiring lesser dosages of antigen. However,the only approved adjuvant for human use in the United States isaluminum hydroxide, also known as alum. Adjuvanted vaccines arepresently prepared by absorbing antigens on alum and then injectingthese materials intramuscularly. Examples of alum adjuvanted vaccinesinclude Diphtheria and Tetanus toxoid vaccines.

A problem with the use of alum in a number of vaccines is that certainalum-antigen complexes can be toxic. This is the exact problem withusing an alum adjuvant for an Influenza vaccine. Present Influenzavaccines are non-adjuvanted, since Influenza absorbed onto alum istoxic. For this reason, although Influenza vaccine has been approved forhuman use since the 1950s, it has never been approved in any type ofadjuvanted form.

Accordingly, an object of the invention is to provide an adjuvantedvaccine which is physiologically safe and effective.

Another object of the invention is to provide an Influenza vaccine usinga starburst dendrimer as an adjuvant.

A further object of the invention is to provide a method of immunizinghumans against Influenza using a starburst dendrimer-adjuvanted vaccine.

These and other objects and features of the invention will be apparentin the following description and the drawings.

SUMMARY OF THE INVENTION

The present invention features a vaccine having a starburst dendrimer asan adjuvant. The preferred vaccine is for Influenza and contains aneffective amount of a composition form ed of an Influenza antigen and astarburst dendrimer in a physiologically compatible carrier. The use ofthe starburst dendrimer makes it possible to adjuvant Influenza withoutproducing a toxic complex since even a small amount of the dendrimeracts as an effective adjuvant. The use of a dendrimer as an adjuvantmakes it possible to use an amount of Influenza antigen which issubstantially reduced from the amount necessary to yield a compatibleantigenic response if the antigen is given without the dendrimer. Thepreferred dendrimer is a poly(amidoamine) dendrimer, preferably aGeneration 3-Generation 10 dendrimer, more preferably a Generation3-Generation 8 dendrimer, with Generation 6 being the most preferreddendrimer. Although any Influenza virus antigen could be used in thecomposition, a composition containing multiple Influenza virus antigenssuch as trivalent split virus Influenza antigens, are preferred. Thephysiologically compatible carrier is preferrably selected from thegroup consisting of distilled water, phosphate buffered saline, normalsaline, and mixtures thereof; however, any other physiologicallycompatible carrier could be used. The starburst dendrimers can be usedin a dilute form, thereby avoiding any potential problems that may stemfrom use in more concentrated form, such as toxic side effects.

The invention further features a method of preparing a vaccine forprotection against illnesses, primarily viral illnesses such asInfluenza. The vaccine used in this method uses a lower level of antigenthan conventional vaccines due to the adjuvant properties of thestarburst dendrimers. Briefly, the Influenza antigen, which may be asingle antigen or a multiple antigen such as a trivalent split virusantigen, is mixed with the starburst dendrimer and the resulting mixtureis diluted with a physiologically compatible carrier. The same type ofdendrimers as described previously are preferably used in this method.

One advantage of the PAMAM dendrimers is their positive charge. ThesePAMAM dendrimers adhere or bind more easily to proteins or othercompounds with negative isoelectric focusing points. Influenza virus,when in antigenic form, has such a negative isoelectric focusing point.Other viruses, or antigenic determinants of viruses, bacteria, fungi, orparasites which have such a negative isoelectric focusing point, canalso be used in the methods of the invention.

Other features of the invention will be readily apparent from thedetailed description.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 of the drawing shows the formation of Generation 0, Generation 1and Generation 2 PAMAM dendrimers by Michael addition of ethylenediamine and methyl acrylate; and

FIG. 2 of the drawing shows the correlation between the number ofprimary amine groups in three generations of dendrimers plotted againstthe mean IFA titer from mice immunized with vaccine adjuvanted withthose dendrimers.

DETAILED DESCRIPTION OF THE INVENTION

The present invention utilizes the adjuvant properties of dendrimers,primarily mid-Generation poly(amidoamine) dendrimers, to provide anantigenic response which is much greater than is otherwise available.The use of these dendrimers allows very low levels of antigen to beused, making this an economically viable and safe method. In addition,the dendrimers are used at a very low level as well, providingprotection against dendrimer toxicity.

For test purposes, an Influenza vaccine was used. This was commerciallyavailable trivalent split virus Influenza vaccine, a subvirion fluvaccine, obtained from Pasteur Merieux. The vaccine is made byinoculating embryonated eggs with virus and aspirating the infectedfluid 3 days later. The Influenza virus is then purified byultracentrifugation, disrupted with detergent to split the virus, andundergoes several additional purification steps before being resuspendedin phosphate buffered saline. While it is not completely clear preciselywhat antigens are involved in the immune response, it is assumed thatthe hemaglutinating antigen on the surface of the virus is one of theprotective antigens.

The following examples show the efficacy of the present invention.

EXAMPLE I

In this example, a commercially prepared trivalent split virus Influenzavaccine was used to show antigenic response and safety. Generation 6PAMAM dendrimers having 256 amidoamine groups (MW 58,048) obtained fromDendritech, Inc. of Midland, Mich., were brought to pH 7.4 by theaddition of concentrated hydrochloric acid. Dendrimers are presentlyavailable suspended in methanol. Before they can be used in the practiceof this invention, the methanol should be removed, e.g., rotoevaporateto dryness, after which the pH should be titrated to neutral withconcentrated HCl. The dendrimers may then be resuspended in apharmaceutically compatible solution, such as H₂ O or PBS. Other methodsof removing the methanol carrier will occur to those skilled in the art,and are encompassed by the methods of the invention. This dendrimersolution was mixed with the trivalent Influenza vaccine at aconcentration of 35 μg/mL, yielding final concentrations of 29 mg/mLPAMAM dendrimer and 21 μg/mL Influenza vaccine. Two 10-fold serialdilutions of the vaccine/dendrimer product were performed and comparedwith similar serial dilutions of the dendrimer alone and the Influenzaantigen alone.

Groups of 6 week old C3H female mice (7-10 mice per group) were injectedwith the dendrimer alone, Influenza antigen alone, or the PAMAMdendrimer adjuvanted-Influenza vaccine. The animals were prebled at day0, and then bled on day 31 after initial immunization. The animals werere-immunized on day 34 after initial immunization and then sacrificedand bled out on day 65. Indirect immunofluorescent assays (IFA) wereconducted on all bleeding samples and hemagglutination inhibition assays(HI) were performed only on the terminal bleeding sample because of thelarge amount of blood needed. The results of all assays are reported asthe reciprocal of the highest titer of antisera detected as positive.Table 2 shows the results of the IFA testing and the HI testing.

As can be seen from Table 3, the undiluted Influenza/dendrimer vaccinewas too concentrated; in fact, it was toxic and all of the animals diedwithin two hours of the first injection. The dendrimer itself, at thesame 2.9 mg/mL concentration, also caused death in the same time period.Accordingly, only diluted form (1:10 and 1:100) data is shown in Table2. As can be seen from Tables 2 and 3, dilute concentrations ofdendrimers have a significant adjuvant effect, with no toxicity.

                                      TABLE 2                                     __________________________________________________________________________    Immunogenicity of G6 Dendrimer-Influenza Vaccine in Mus musculus strain       C3H                                                                                                             Amount of                                                                            Pre-bleed                                                                           Day 34                                                                             Final Day                                            Amount HA                                                                            Dendrimers                                                                           Reciprocal                                                                          Reciprocal                                                                         Reciprocal                                                                          Final Day           Murine Strain                                                                        IFA Antigen: A/Texas H1N1                                                                         in Micrograms                                                                        in Milligrams                                                                        IFA   Mean IFA                                                                           Mean                                                                                Mean                __________________________________________________________________________                                                              HI                  C3H    Pasteur Merieux Subvirion Flu PBS mixed - IM                                                      1.7    0      0     NP    240   56                 10/group                                                                             (Influenza Ag alone)                         (day                                                                                (day 42)            C3H    PM Subvirion Flu PBS mixed w/Dendrimers,                                                          0.21   0.29   0     2586 4096  1680                10/group                                                                             pH = 7.4, diluted 1:10                       (day                                                                                (day 42)                   (Influenza and G6 Dendrimers)                                          C3H    PM Subvirion Flu Diluted 1:10 with PBS                                                            0.17   0      0      36   176   41                 8/group                                                                              (Influenza Ag alone)                         (day                                                                                (day 65)            C3H    PM Subvirion Flu PBS mixed w/Dendrimers                                                           0.021  0.02   0      109  448   256                10/group                                                                             pH = 7.4, diluted 1:100                      (day                                                                                (day 65)                   (Influenza and G6 Dendrimers)                                          C3H    PM Subvirion Flu Diluted 1:100 with PBS                                                           0.017  0      0        0.6                                                                              20   NP                  7/group                                                                              (Influenza Ag alone)                    (2/7 resp.)                                                                        (6/7                                                                                (day 65)                                                                (day                      __________________________________________________________________________                                                        65)                        Volume of Vaccine Innoculum per mouse 100 μLIM                             NP = not performed                                                       

                  TABLE 3                                                         ______________________________________                                        Toxicity of G6 Dendrimer-Influenza Vaccine in Mus musculus strain C3H         Murine Strain                                                                           IFA Antigen: A/Texas H1N1                                           ______________________________________                                        C3H       Dendrimers = 7.4 (G6) undiluted (29 mg/mL)                          10/group  (All animals died within 2 hours of injection)                      C3H       PM Subvirion Flu PBS mixed w/Dendrimers                             10/group  pH = 7.4 (G6) undiluted                                                       (All ten animals died within 2 hours of injection)                  C3H       Pasteur Merieux Subvirion Flu PBS mixed - IM                        10/group  (All ten animals alive at day 42 after 2 injections)                C3H       PM Subvirion Flu PBS mixed w/Dendrimers                             10/group  pH = 7.4 (G-6) diluted 1:10                                                   (All ten animals alive 65 days after 2 injections)                  C3H       PM Subvirion Flu Diluted 1:10 with PBS                              8/group   (All eight animals alive 65 days after 2 injections)                C3H       PM Subvirion Flu PBS mixed w/Dendrimers                             10/group  pH = 7.4, diluted 1:100                                                       (All ten animals alive 65 days after 2 injections)                  C3H       PM Subvirion Flu Diluted 1:100 with PBS                             7/group   (All seven animals alive 65 days after 2 injections)                ______________________________________                                         Volume of Vaccine Innoculum per mouse 100 μLIM                        

Comparison of the 1:10 antigen/dendrimer vaccine with the same amount ofvaccine alone shows the effectiveness of the invention. After only oneinjection, the non-adjuvanted vaccine had a titer of 36 while theadjuvanted had a titer greater than 2500, a 71-fold increase. Similarlyafter the second injection, the values are 176 and 4,096, respectively,an improvement of over 23 fold. The results at the higher dilution, the1:100 dilution, are just as dramatic. After a single injection, there isa 181-fold greater response from the adjuvanted vaccine and after thesecond injection, there is better than 22-fold increase in response. Infact, the value after two injections with 100 fold dilution of theantigen/dendrimer vaccine antigenic response than the 1:10 dilution ofthe antigen alone.

The hemagglutionation inhibition assay (HI) yield similar results.Undiluted Influenza antigen yielded a titer of 56 while theantigen/dendrimer combination yielded titers of 1680 and 256 at 1:10 and1:100 dilutions, respectively. A titer of 32 is normally considerednecessary for effectiveness.

In light of the foregoing, it is clear that the dendrimers act asexceptional adjuvants, even on materials that are notoriously difficultto adjuvant.

EXAMPLE II

comparison of the adjuvanticity of three generations of dendrimers, G0,G3 and G6, prepared and administered as described in Example I above, indilutions of 1:10 and 1:100, shows a correlation between the number ofprimary amine groups and the resulting mean IFA titer in mice. This datais summarized in Table 4, and illustrated graphically in FIG. 2. As canbe seen from Table 4, an adjuvant effect is not produced with G0dendrimers, while an effect is visible, but low, when G3 dendrimers areused. Dendrimers of Generation 6 are clearly effective adjuvants. As canbe seen from FIG. 2, adjuvant effect correlates, in a substantiallylinear fashion, with the number of primary amine groups in eachgeneration.

                  TABLE 4                                                         ______________________________________                                        Summary of Dendrimer Structure-Influenza                                      Primary Immunization IFA Data in Mice                                                                    Number of                                                                            Mean  Mean                                                             Primary                                                                              H1N1  H1N1                                           Dendrimer                                                                              Molecular                                                                              Amine  IFA   IFA                                   Vaccine  Category Weight   Groups at 1:10                                                                             at 1:100                              ______________________________________                                        G-0/TFlu PM                                                                            0         517      4      134   15                                   TFlu PM alone                                                                          NA       NA       NA      47    17                                   G-3/TFlu PM                                                                            3         6909     32    *448  *154                                  TFlu PM alone                                                                          NA       NA       NA      47    17                                   G-6/TFlu PM                                                                            6        58048    256    *2586 *109                                  TFlu PM alone                                                                          NA       NA       NA      36      0.5                                ______________________________________                                         NA--Not Applicable                                                            * = Adjuvant Effect                                                      

The foregoing example and description is merely illustrative of theinvention. The invention is defined by the following claims and thoseskilled in the art will recognize there scope and the scope ofequivalents thereof.

What is claimed is:
 1. A vaccine for Influenza comprising an effective amount of a composition formed of an Influenza antigen and a starburst dendrimer in a physiologically compatible carrier; the amount of said Influenza antigen in said composition being substantially reduced from the amount necessary to yield a comparable antigenic response if given without said dendrimer.
 2. The vaccine of claim 1 wherein said dendrimer comprises a poly(amidoamine) dendrimer.
 3. The vaccine of claim 2 wherein said dendrimer is selected from the group consisting of Generation 3-Generation 10 dendrimers.
 4. The vaccine of claim 3 wherein said dendrimer is selected from the group consisting of Generation 5-Generation 8 dendrimers.
 5. The vaccine of claim 4 wherein said dendrimer is a Generation 6 dendrimer.
 6. The vaccine of claim 1 wherein said Influenza antigen comprises multiple Influenza virus antigens.
 7. The vaccine of claim 6 wherein said multiple antigens comprise trivalent split virus Influenza antigens.
 8. The vaccine of claim 1 wherein said physiologically compatible carrier is selected from the group consisting of distilled water, phosphate buffered saline, normal saline, and mixtures thereof.
 9. A method of preparing a vaccine for protection against Influenza which requires a lower level of antigen than conventional Influenza vaccines, the method comprising the steps of mixing an Influenza antigen with a starburst dendrimer, and diluting the mixture with a physiologically compatible carrier until said vaccine is prepared.
 10. The method of claim 9 wherein said dendrimer comprises a poly(amidoamine) dendrimer.
 11. The method of claim 10 wherein said dendrimer is selected from the group consisting of Generation 3-Generation 10 dendrimers.
 12. The method of claim 11 wherein said dendrimer is selected from the group consisting of Generation 3-Generation 8 dendrimers.
 13. The vaccine of claim 12 wherein said dendrimer is a Generation 6 dendrimer.
 14. The method of claim 9 wherein said Influenza antigen comprises multiple Influenza virus antigens.
 15. The method of claim 14 wherein said multiple antigens comprise trivalent split virus Influenza antigens.
 16. The method of claim 9 wherein said physiologically acceptable carrier is selected from the group consisting of distilled water, phosphate buffered saline, normal saline, and mixtures thereof. 